Interpretive Summary: Insects possess highly effective immune defense systems, which has in part contributed to their ability to survive within highly diverse niches. An important step for any defense system is to first recognize foreign surfaces or “non-self” material and activate an appropriate response. Insects (like vertebrates) use proteins called pattern recognition proteins that initiate various downstream immune responses and provide protection from potential pathogenic or opportunistic organisms. In previous work, we showed that a pattern recognition protein called beta-1,3-glucan recognition protein or beta-GRP from Plodia interpunctella, a serious pest of stored products, functioned to recognize components that make up microbial surfaces and activated insect immune responses. Here, we determined the three-dimensional structure of a portion of beta-GRP and demonstrated that it binds to and forms a large complex of protein and sugar commonly found in microbes. The results suggest that this protein complex may be important for amplification of the initial signal required to mount a defense against potential pathogenic organisms. These results represent new information into understanding how insects mount immune responses and could be exploited for novel control of insect pests.

Technical Abstract:
In response to invading microorganisms, insect beta-1,3-glucan recognition protein (beta-GRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades associated with the prophenoloxidase (proPO) and Toll pathways; it also agglutinates bacterial and fungal cells. The amino-terminal domain of beta-GRP (N-beta-GRP) from Plodia interpunctella is sufficient for beta-1,3-glucan binding, and in the presence of laminarin, a water-soluble beta-1,3-glucosidic oligosaccharide, N-beta-GRP significantly increases proPO activity. Here we report on solution biophysical studies, including NMR and analytical ultracentrifugation (AUC), of the interaction between N-beta-GRP and laminarin. The results indicate that while the protein and the ligand exist as monomers, complex of N-beta-GRP and laminarin readily undergoes self-association upon ligand-binding. Formation of a water-soluble N-beta-GRP:laminarin complex results in the loss of NMR signals from the backbone 15N-1H groups of the protein. AUC studies indicate that this protein:carbohydrate macro complex (~ 95 kDa) has a high stability constant, as it does not dissociate upon dilution to submicromolar concentrations. These results are in contrast to a recent report that proposes that N-beta-GRP binds to a triple helical structure of laminarin on the basis of an X-ray crystallographic study of the complex formed between P. interpunctella N-beta-GRP and the hexasaccharide, laminarihexaose [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., and Yamaguchi, Y. (2011) J. Biol. Chem. 286, 29158-29165]. We further demonstrate by site-directed mutagenesis experiments and prophenoloxidase activation measurements that electrostatic interactions between the protein molecules contribute to the stability of N-beta-GRP:laminarin complex and a decrease in stability causes a reduction in the activation of the proPO pathway. These novel findings suggest that ligand-induced self-association of beta-GRP:beta-1,3-glucan complex is a means of amplification of the initial signal of pathogen recognition for activation of immune response cascades.